CN104737001B - The method of the LIBS quantitative measurments of bio-molecular target on biochip - Google Patents
The method of the LIBS quantitative measurments of bio-molecular target on biochip Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/71—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited
- G01N21/718—Laser microanalysis, i.e. with formation of sample plasma
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/71—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited
- G01N21/714—Sample nebulisers for flame burners or plasma burners
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/71—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited
- G01N21/73—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited using plasma burners or torches
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/061—Sources
- G01N2201/06113—Coherent sources; lasers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4731—Casein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
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- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
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- Food Science & Technology (AREA)
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A kind of method for being used for quantitative analysis by plasma emission spectroscopy, the plasma is the plasma of at least one target (201) being included on biochip induced by laser beam, it is characterized in that methods described includes the use of adjuvant (202), the adjuvant allows to be formed can be with least one target (201) while the dry matrices (203) being ablated to, the dry matrices (203) are configured to improve the analytical property of plasma, the adjuvant (202) has emission spectrum, the wavelength that the emission spectrum spectral line of adjuvant has is different from the wavelength of the optic spectrum line of the quantitative analysis at least one target (201).
Description
Technical field
The present invention relates to the method and apparatus of the quantitative measurment of bio-molecular target on biochip.It especially suitable for
The field of proteomics, and more specifically it is applied to the protein phosphorus via laser induced plasma emission spectrum technology
The quantitative measurment of acidifying.
Background technology
In proteomics field, effort is directed to identifying protein and their own present in cell extract
Concentration, and it is directed to identifying the posttranslational modification of these protein, this represent their activity.Most feel under this background
A kind of posttranslational modification of interest is phosphorylation：Protein phosphorylation is from the angle of cognition in modern biology field or from controlling
It is basis for diagnosis angle in treatment field.This function in many bioprocess (adjust by such as epigenetic regulation, nutrition
Control, DNA are repaired, hormone control etc.) it is actually essential.Made it possible to without known simple and direct technology
Identify the protein in Biomedia and assess its phosphorylation degree.
One known analytical technology by the way that protein extract is placed in the array on holder in being arranged
(each put and be made up of multiple identical probes) to analyze the protein extract on " point " position, the array on the holder leads to
Frequently referred to biochip or microarray.The target found in protein extract to be analyzed, the spy are identified with specific probe
Specific probes can be antibody, acceptor or any other point specifically associated with one of given protein or its modification
Son.In the case of antibody, holder is referred to as " antibody biochip ".Therefore can be simultaneously or several by suitable analytical technology
Simultaneously analyze multiple target biomolecules.Various technologies make it possible on antibody biochip carry out therefore retain or
The actual analysis of " isolation " element.
Analysis for the phosphorylation of nucleic acid on biochip, a kind of known technology be by induced with laser etc.
Emission Spectroscopy in gas ions carries out this analysis, the commonly referred to as abbreviation corresponding to term LIBS
LIBS.The technology includes the sample isolated by laser beam ablation in biochip surface and including producing plasma, should
Then plasma is analyzed by spectroscopic methodology.Transmitting for each laser, can be with the hair of the chemical element of Record analysis
Penetrate spectrum, and the focusing coordinate of the laser beam of record sample surfaces.Then suitable computational methods can make it possible to build
The foundation drawing of vertical sample surfaces.Such as it is used for being described in patent application disclosed in bibliography FR 2 929 011 in biology
Analyze the apparatus and method of the quantitative measurment of bio-molecular target present on holder.Such as with bibliography FR 2 964 458
LIBS analytical equipments are described in disclosed patent application.
However, there are multiple problems in the LIBS analyses of the protein phosphorylation on biochip in principle.First
Problem is related to the presence of external source phosphorus, and it has covered the signal found.Second Problem is usually used with being used for bioanalysis
Holder and LIBS are related the fact that analysis is incompatibility, because the plasma formed on this type supports can not
Analyzed under acceptable terms.3rd problem and biomolecule (such as protein) have very small amount of in its structure
The fact that phosphorus, is related, therefore it is difficult to detect by LIBS.4th problem and the signal that obtains can not be with readily can models
The fact that the mode of change changes (such as linear) as the function for the amount found is related：In other words, for quantification of protein
Signal normalization is problematic.
The content of the invention
The purpose of the application is by proposing for the method by LIBS technology quantitative analysis bio-molecular targets and setting
It is standby at least to overcome the shortcomings that above-mentioned.The present invention uses suitable holder and method, it is allowed to which formation is easily analyzed by spectrum
Plasma, the abundance of the amount of searching is proportional to by the LIBS signals of plasma emission.
Therefore, the theme of the present invention is the method and apparatus for quantitative measurment.More specifically, one of the present invention
Theme is the method for being used for quantitative analysis by plasma emission spectroscopy, and the plasma is included by what laser beam induced
The plasma of at least one target on biochip, it is characterised in that it includes the use of adjuvant, so that forming energy
Enough dry matrices being ablated to simultaneously with least one target, configure the dry matrices to improve the analytical of plasma
Matter, the adjuvant have emission spectrum, the wavelength that its emission spectrum spectral line has and quantitative point at least one target
The wavelength of the optic spectrum line of analysis is different.
In one embodiment of the invention, at least one target can be by least one probe in biochip
Upper isolation, so as to form probe-target complex between each probe and corresponding target.
In one embodiment of the invention, the use of the adjuvant can allow the dry matrices of amorphous structure
Formed.
In one embodiment of the invention, the use of the adjuvant allows the formation of the dry matrices of crystal structure.
In one embodiment of the invention, dry matrices can be formed to cause probe-target complex in holder
Surface can use, and probe-target complex dry matrices as described in certain amount wrap into whole or in part.
In one embodiment of the invention, dry matrices and probe can be fixed to holder.
In one embodiment of the invention, the dry matrices that adjuvant molecules are formed can be directly with compound to spy
Pin.
In one embodiment of the invention, the dry matrices that adjuvant molecules are formed can be fixed to holder, probe
Adjuvant molecules are fixed to, adjuvant molecules are fixed to holder.
In one embodiment of the invention, one layer can be formed by placing one layer of adjuvant in support surface
Dry matrices, then probe is placed on the surface of the dry matrices in the support surface being consequently formed.
In one embodiment of the invention, the chemical combination that dry matrices can be by absorbing wavelength close to the wavelength of laser beam
Thing is formed, so as to which the wavelength that laser uses is included in the absorption spectrum of dry matrices.
In one embodiment of the invention, adjuvant can include water and the solution from sugar, polysaccharide and disaccharides composition.
In one embodiment of the invention, each probe can usually be marked with the standardization member for forming internal standard
Note.
In one embodiment of the invention, each probe can usually be marked by grafting by standardization member.
In one embodiment of the invention, standardization element can be made up of boron.
Subject of the present invention further relates to the equipment for including the holder containing multiple sites, and each site includes multiple spies
Pin, probe graft to the adjuvant molecules for allowing dry matrices to be formed simultaneously.
In one embodiment of the invention, the holder can include organic compound, monolithic compound or have
Machine diamond compound.
Subject of the present invention is further related to for carrying out the solution according to the described embodiment method of any one, and it is special
Sign is that it includes the adjuvant for allowing dry matrices to be formed.
In one embodiment of the invention, the solution for progress according to the method for one of described embodiment
The element to the optical signalling for being used to standardize the plasma by laser beam induction of certainty ratio can be included.
Another advantage provided by the present invention is the phosphorus content in its permission biomolecule (such as protein) and mixture
Simple, quick, quantitative and vector analysis.
According to the present invention, quantitative analysis comprising with arrangement target and/or probe array biochip on carry out.
The point of each probe can identify and isolate the target biomolecule being present in mixture to be analyzed.According to one of the present invention
Specificity, it is proposed that allow the formation of dry matrices using adjuvant, the matrix is closely connected with target or probe-target complex.Art
Language " close connection " is being supported this means dry matrices, wrapped into or around target or probe-target complex, so that target or spy
The laser ablation of pin-target compound and by LIBS on the point of Biochip arrays caused plasma except being given birth to comprising target
Outside thing molecule or target-probe complex, also comprising adjuvant molecules and/or its element decomposed.Below, it is believed that in biochip
Dry matrices are formed in the form of solid chemical compound crystallize or amorphous after drying.As infinite embodiment, dry
Matrix can be the form of adduct (for example, the mixture of probe, target and adjuvant molecules, the crystal of mixing or amorphous or bag
Wrap up in the mixture of target and/or probe).The various configurations of dry matrices are entered hereinafter as embodiment on biochip holder
Row description, reference picture 2A to 2D.It should observe that method and apparatus according to the invention can also be applied to be included in holder
The biochip of upper deposition or multiple targets of isolation, it is not necessary to associated with probe.
Adjuvant is especially selected so that its substantially non-phosphatization, so as to which it is denatured Ag-Ab key.This anticipates first
The emission spectrum that taste the molecule for forming adjuvant and matrix itself is different from the spectral line of emission that quantitative phosphorus uses, and this is adapted to mark
Standardization signal, secondly this means hybridization of the adjuvant with target and/or probe-target complex or compound phase are compatible.
Adjuvant for forming dry matrices may, for example, be soluble.The following describe the implementation of suitable solution
Example.Also select dry matrices improves the analysis for being used for spectral technique analysis by laser-produced plasma so as to further have
The advantages of characteristic.Especially, dry matrices can by its absorbing wavelength close to the wavelength of laser beam used compound come
Formed, so as to which the wavelength that laser uses is included in the absorption spectrum of dry matrices.
Brief description of the drawings
Other features and advantages of the present invention as the description that embodiment provides will become obvious reading, on accompanying drawing its
Represent：
- Fig. 1, show the flow chart of the analysis method according to a specific embodiment of the invention；
- Fig. 2, show a specific embodiment according to the present invention, the analysis on the biochip including probe/target
The schematic diagram of method General Principle；
- Fig. 3 A, 3B, 3C and 3D, show the general of analytical equipment in various configurations according to a particular embodiment of the invention
Including property schematic diagram；
- Fig. 4 A and 4B, show the analytical equipment for also including the configuration Plays element for corresponding respectively to Fig. 3 A and 3C
Broad schematic.
Embodiment
Reference picture 1, it can be included preparing biochip according to the analysis method of any infinite embodiment of the present invention
First step 101.Can be the second step 102 analyzed by LIBS after first step 101, during second step 102
According to techniques known in themselves, significantly essentially formed on target biomolecule to be analyzed by way of laser ablation
Gas ions.
Preparation process 101 can include the first sub-step and the second sub-step, and first sub-step is included in holder
Upper deposit biomolecules (such as independent target and/or probe), are then dried；Second sub-step can include, in office
After the saturation sub-step of choosing, isolate multiple target biomolecules on the support, be optionally incorporated into corresponding thereto
On antibody, probe substrate is formed.Therefore each target biomolecule is fixed to identifies its probe on holder.
During first step 101, according to the specificity of the present invention, the formation of matrix is also dried.What is formed is dry
Dry matrix makes it possible to ensure preferably sharp by the emission spectrum of laser-produced plasma during second step 102
The property used.The formation of dry matrices with target or with probe-target complex so as to melting simultaneously.Therefore, target interested is targetted
The transmitting of the ablation laser of biomolecule ensure that the ablation of adjacent dry matrices part.Therefore, can arrange dry matrices from
And completely or partially encase target biomolecule or probe-target complex.Dry matrices can also be arranged in target biology point
Below son or probe-target complex, on condition that the near-end of dry matrices is enough to be used in target biomolecule or probe-target complex
Laser emission also melt neighbouring dry matrices part.Describe to 3D referring to Fig. 3 A and supported relative in biochip
The various possible configurations of the target biomolecule or the dry matrices of given probe-target complex that are given on thing.
Dry matrices are also with so as to allowing the analytical property for improving LIBS plasmas to configure.In addition, the matrix should not
Make probe-target identification denaturation (in the appropriate case), and its own should not contain phosphorus.
Dry matrices can be amorphous or crystal, and can be formed by way of adjuvant.Adjuvant can example
Soluble adjuvant in this way.
In fact, in a specific embodiment, for the analysis method according to one of described embodiment
Solution, include the part of biomolecule to be analyzed, the adjuvant for allowing dry matrices to be formed can also be included.In some implementations
In scheme, adjuvant can be incorporated at least one solution used for biochip with target protein hybridization or for hybridizing
In the solution that rinsing biochip uses afterwards.In this embodiment, after compound and/or rinsing, adjuvant allows in biological core
Dry matrices are formed on the surface (particularly in each point) of piece.
In other embodiments, formed dry matrices adjuvant can on the support deposition probe the step of during,
Introduced when manufacturing biochip：Either by being introduced in deposited probes solution or being after the deposition of probe, lead to
Cross chemical method and be directly grafted adjuvant molecules to the surface for being used to fix the holder that probe uses.For example, sucrose molecule
It can be grafted on the surface of biochip, with thionyl chloride (SOCl in a manner of its alcohol functional group2) activate.
In another embodiment, adjuvant molecules can be grafted to first on the surface of biochip holder and so
Be activated (if being needed as the functional group of adjuvant properties) afterwards, and the side that probe can pass through adjuvant molecules as point
Formula is grafted on the surface of biochip.
Advantageously, at least one rinsing or hybridization solution can include metal or halo standardization element, and it is launched
LIBS signals it is different from the LIBS signals that phosphorus is launched and proportional with the abundance of the metallic element.Especially, Ke Yixuan
Select the standardization element so that it is used for the selected spectral line of emission of phosphorus analysis identical with the function as use device
Spectral window in.Preferably, the spectral line of emission can be different in terms of the spectrometer used, i.e., enough for given spectrometer
Far, so as to distinguish them.
Generally, standardization element can be incorporated into the adjuvant for allowing dry matrices to be formed.
It can be such as boron to standardize element, take separation, be incorporated into molecule or capture in molecule cage or crypts,
And reproducibly combined with target.
Advantageously, standardizing element such as boron can be directly compound to probe antibody, or optionally compound to target.
The implementation of the configuration of the standardization element relative to target molecule and/or probe is described referring to Fig. 4 A and 4B
Example.
The holder for forming biochip can be without phosphorus carbon-based holder, and it includes organic compound, i.e., elemental carbon,
Any combination of hydrogen, oxygen and nitrogen.The holder for forming biochip can be monolithic (such as diamond), glass or organic
Diamond, it promotes the formation that can utilize plasma under good conditions.Its branch that can be made up of monolithic nano particle
Hold thing, the monolithic nano particle covered with deposition or the probe that is grafted on vitreum or organic support materials.
The adjuvant itself that permission dry matrices presented hereinbefore are formed is not configured to be denatured probe-target key and not wrapped
It is phosphorous.Adjuvant can be formed for example by polysaccharide (such as sucrose) or not phosphorous any other polysaccharide.
Above-mentioned second step 102 allows to be analyzed by LIBS type spectrum using suitable equipment, and
The sub-step of itself known method can be included.The second step 102 includes the analytical equipment for passing through LIBS spectrum
Use, the device for being used for launching intense pulse laser bcam or " ablation beam " is especially comprised at least, for moving sample to be analysed
Device, record the device and computing device of detection.
Fig. 2 is the schematic diagram of the general principle for the analytical procedure for illustrating a specific embodiment according to the present invention.
According to the general principle of the present invention, when it is applied to target protein, multiple target proteins 201 can be used as more
Individual spot deposition is on the holder 200 of biochip.In the embodiment illustrated in figure 2, holder 200 can also be known including multiple
The probe 211 of other target protein 201.Include the use of adjuvant 202, such as soluble assistant according to the analysis method of the present invention
Agent, it allows the formation of dry matrices 203, and it encases probe-target complex completely in the embodiment illustrated in figure 2.Therefore exist
After first step 101 above-mentioned on described by figure 1 above, in the embodiment of the unrestricted explanation of the present invention, bag
The phase of second step 102 that containing multiple target proteins 201 and also the biochip comprising multiple probes 211 can be generally noted above
Between carry out LIBS analyses and be referred to Fig. 1 description.
Fig. 3 A and 3D show the broad schematic of the analytical equipment according to various embodiments of the invention, corresponding to drying
The various configurations of matrix and biochip holder.
In the embodiment illustrated by Fig. 3 A, it corresponds to target biomolecule, corresponding probe and in biological core
First configuration of the dry matrices on piece holder, each target biomolecule 201 being fixed on probe 211 can be by certain
Quantity dry matrices 203 are encased, and the probe 211 is arranged on the holder 200 of biochip and identifies each target biology
Molecule 201, the dry matrices 203 are formed by previously described suitable adjuvant.Probe-target biological molecular bond 211,
201 fully can encase (embodiment as illustrated in FIG.) by dry matrices or only partially wrap in dry matrices.
According to the embodiment illustrated by Fig. 3 B, it corresponds to the second configuration, on the holder 200 of biochip
Probe is placed, the probe is used for forming probe-target biological molecular bond 211,201, the holder 200 of the biochip
Can on its surface with dry matrices 203 come saturation.According to the second configuration, such as biochip can be placed on high-ranking officer's oxidant layer
Holder 200 surface on, the holder 200 of the biochip has been covered with probe-target biological molecular bond 211,
201.This embodiment can have the advantage that the whole surface of the holder 200 of biochip may be used in terms of ease for use
Dry matrices 203 carry out saturation.
Embodiment according to being illustrated by Fig. 3 C, it corresponds to the 3rd configuration, is placed on the holder 200 of biochip
Each probe-target biological molecular bond 211,201 can wrap in dry matrices 203, formed dry matrices 203 adjuvant
May be directly compound to probe 211.
According to the embodiment illustrated by Fig. 3 D, it corresponds to the 4th configuration, can be by placing or being grafted one layer
Adjuvant forms one layer of dry matrices 203, such as the formation uniformly over the surface of the holder 200 in biochip, Ran Houfang
The probe 211 put directly is grafted in matrix.Target biomolecule is directly compound or is hybridized on dry matrices corresponding thereto
The probe answered.The pattern of replacement can be included on grafting probe to holder, then the molecule of graft bases on the support, from
And therefore surround target.4th embodiment is in terms of industrial point of view with sizable advantage.Specifically, can supply including biological core
The kit of piece, the layer of dry matrices 203 can be formed from the holder of biochip, such as the biochip of routine.Operation
Then probe-target biological molecular bond can normally be placed on the surface of this biochip by person in the usual manner
On.
Subject of the present invention is also for the prehybridization of biochip or the equipment of hybridization or " kit " for example, at least one
Kind includes the solution of adjuvant, and it is used for the prehybridization of biochip, hybridization or rinsing, and the adjuvant allows the table in biochip
Dry matrices are formed on face.
Therefore, subject of the present invention is also biochip, therefore the holder of the biochip is included in its surface
One layer of dry matrices 203.
No matter the embodiment considered, the thickness of the dry matrices 203 advantageously formed on target biomolecule are averaged
1 millimeter is not should be greater than.For bigger thickness, it may not actually be included by laser-formed plasma and be possibly used for analyzing
Rational proportion in trace target biomolecule.
In the embodiment of each description, each probe 211 can be advantageously comprising the standardization for forming internal standard
Element, it makes it possible to the signal that standardization is obtained by LIBS.According to a feature of the present invention, such as previously retouch
State, standardization element can usually be formed by the member of directly compound to each probe 211, such as pass through crosslinking technology.Such as
Fruit probe 211 is formed by antibody, therefore the antibody can be marked usually directly through grafting with standardization member.Standardization
Element for example can be made up of element (such as boron).
The embodiment of the standardization element arrangements relative to target molecule and/or probe is described referring to Fig. 4 A and 4B,
It corresponds respectively to the configuration described by previous 3A and 3C.
Reference picture 4A, standardization element 405 can be attached directly to the probe 211 on holder 200, such as logical
Cross crosslinking technology.Configured according to corresponding to first with reference to previously described Fig. 3 A, the group formed by probe 211 and target 201
Part can be encased by dry matrices 203.
Reference picture 4B, standardization element 405 can be directly fixed to the probe 211 on holder 200, such as via
Crosslinking technology.According to can be with corresponding to the adjuvant molecules for reference to the previously described configurations of Fig. 3 C the 3rd, forming dry matrices 203
It is such as directly compound to probe 211 with the embodiment.
The wavelength that the optic spectrum line of adjuvant has is different from the wavelength for optic spectrum line used in quantitative analysis, and it is wrapped
Include for the optic spectrum line of standardization and for quantifying target and/or the optic spectrum line of its posttranslational modification.
Two proposals of the method one of according to an embodiment of the present invention carried out by applicant described in detail below
Condition.Applicant has formulated the condition of analysis method especially by the analysis for carrying out phosphorylating protein, more specifically in A2
Analyzed on the external model of beta-casein reconstruct, wherein qualitative known by the analysis method of inductive emission spectrum
The solution of the casein of concentration, the solution is generally referred to as Inductively Coupled Plasma Atomic Emission Spectrometry and (corresponds to abbreviation
ICP-AES)；And analyzed on the biomolecule model containing external source phosphorus, wherein the journey of molecular target H2AX phosphorylations
Degree can be induced arbitrarily.According to the non-limiting embodiment of the present invention, the side related to the analysis is described in more detail below
Method and equipment, the analysis method and equipment can apply to other target biomolecules, the albumen of particularly any phosphorylation.Should
It was observed that the embodiments described below is not limit the invention in any way, and embodiment corresponds to particular case, wherein
Adjuvant is added during each method and step, and according to the embodiment with reference to previously described Fig. 2A in biochip
Dry matrices are formed on holder to encase target biomolecule.However, can with reference to the various of Fig. 2 B to 2D described above
The mode of replacement nondistinctive can also be applied and cause similar result.Assistant may also be introduced only in single method step
Agent.
The embodiment (no probe proteins matter chip) of deposition targets on biochip.
The molecular weight of A2 beta-caseins is equal to 23.983 kilodaltons.This albumen can be supplied for example with lyophilized form,
Then, for example, being purified by being commonly abbreviated as HPLC (corresponding to high performance liquid chromatography) commonsense method, such as bag is used
Aqueous (H2O), the solution of trifluoroacetic acid (TFA) and formonitrile HCN (or acetonitrile).The concentration of protein can for example pass through ultraviolet spectrometry
Photometric method determines.
In order to assess the efficiency by LIBS spectrum analyses, the analysis of beta-casein phosphorylation can be by known and good
Reliable alternative is commented to carry out so as to provide reference measure.Therefore, the analysis method of ICP-AES classes, this method can be utilized
It is the reference method for determining the main composition of liquid compound.The applicant is with 0,4,12 and 20 μM of (micromole) concentration
β-casein solution carries out ICP-AES analyses.
In order to determine the sensitivity of the LIBS spectrum quantitative for protein phosphorylation, different amounts of beta-casein is directly
It is deposited on by made of kapton-epoxy resin on holder, kapton-epoxy resin thin film (such as use alpha-cyanoacrylate
Ester adhesive) it is bonded on microslide (such as in the form of 7.5 × 2.5 centimetres of slides).
Thus obtained beta-casein solution is serially diluted to 0 in pH is 7.5,0.1M tris cushioning liquid, 40,85,
170th, 257 and 350 μM of concentration, said preparation are referred to as " preparation A ", including the buffering that pH is 7.5,0.1M tris and 1mM sucrose
The preparation diluted in solution, be referred to as " preparation B ", and be 7.5,0.1M tris, 1mM sucrose and 0.5mM boron comprising pH
The preparation diluted in cushioning liquid, it is referred to as " formulation C ".These preparations are individually repeatedly deposited on kapton-epoxy 5 times
On resin film, and it is dried at room temperature for 30 minutes.
The embodiment of antibody biochip
It can be separated by gradient cell from the human blood sample of test tube of hepari and separate lymphocyte.Cell is delayed with phosphate
Rush salt solution or PBS is washed twice, in culture medium (such as containing 10% hyclone or FCS, Jie of 1% Pen .- Strep
Matter) in be diluted to 106The concentration of individual cells/ml, and immediately with 60Coradiation device with 2Graymin-1Dosage level,
Radiated with 0,0.5,2 and 4Gray.After radiation, cell is rapidly in the medium with 5 × 105Cell/ml concentration dilutes,
And γ H2AX phosphorylation levels it is quantitative before, trained in 37 DEG C of temperature in the atmosphere of the carbon dioxide containing 5% concentration
Support one hour.
In order to obtain the quantitative matrix of phosphorylation for being applied to analyze by LIBS, it is necessary to make antibody matrix in itself without
Phosphorus.Anti- γ H2AXser139Antibody can select for this purpose.Then by the antibody deposition of dialysis by kapton or Kapp
- holder that is formed of epoxy resin on, such as include the holder of 25 microns of kapton layer, or comprising with 25 microns of ring
The holder of 25 microns of kapton layer of oxygen tree lipid layer covering.
Antibody is positioned on kapton-epoxy resin thin film layer.The anti-γ H2AX of 100 microlitres of volumesser139In pH
For in 7.5 cushioning liquid comprising 0.1M tris and 1mM sucrose, in the dialysis four hours of 4 DEG C of temperature.
After dialysis, anti-γ H2AXser139Adjusted in 1mM of the concentration of solution in 0.1M tris cushioning liquid boron to
0.5mg/ml, and on kapton-epoxy resin thin film in each hole of matrix, then the solution of 1 microlitre of volume is positioned over
It is dried at room temperature for 30 minutes.In order to avoid the non-specific binding of protein and kapton-epoxy resin thin film layer, medium
Free epoxide functional groups surpass blocking buffer in the solution of the dextran comprising tris, 1mM sucrose and 5% concentration with 1X
Or SBB solution saturation 30 minutes at room temperature, it is gently mixed.Holder can use the sucrose comprising 1mM and 0.1% poly- sorb
Washed three times in the trsi cushioning liquid of alcohol ester 20 or the solution of 1X TBS types, the solution is also used for washing according to present invention side
Antibody matrix in method.Lasting fixation can be analyzed antibody by Fluorescence Scanner on the support, and phosphorus is not present
It can be confirmed by LIBS.
In order to allow the extraction of target antigen, by with 10000 revs/min of two minutes precipitation lymphocytes of centrifugation, putting
Put in micro-wave oven (450 watts of power) 15 seconds, be then suspended in the protein concentration of 1 mg/ml comprising 0.1M
In the cushioning liquid of tris and 1mM sucrose.
Combined for protein-antibody, protein is diluted to 0.1 milli in the 1X SBB cushioning liquid comprising 1mM sucrose
The concentration of grams per milliliter, and in the culture 12 hours of 4 DEG C of temperature.Resulting antibody matrix in the sucrose comprising 1mM and
Washed in tris (1X TBS) buffer salt solution of 0.1% polysorbate20, and in the tris (1X comprising 1mM sucrose
TBS 15 minutes) are washed in buffer salt solution with stirring, twice, is then dried at room temperature for.
On the analysis by reference to method, flow cytometer is passed through with the lymphocyte aliquot of each dose
Or FACS is analyzed.Cell is not added with hot wash at a temperature of 4 DEG C and washed, then fixed and saturating in the alcohol of 70 ° of normal intensities
Change, continue 1 hour.Then cell is at room temperature with anti-γ H2AXser139Antibody-solutions cultivate one in the PBS comprising 2%FCS
Hour.Precipitated after washing and centrifuging, precipitation is resuspended and is incubated one hour with anti-mouse secondary antibody, it is described anti-
Anti-mouse secondary antibody is marked with the fluorogen or FITC of fluorescein isothiocynate type.Cell is washed out, centrifuges simultaneously settling flux
In the PBS comprising propidium iodide and ribalgilase.Use flow cytometry analysis at least 104Individual cell and each thin
The fluorescence intensity of born of the same parents is determined by suitable analysis software.Then can from analysis cell median fluorescence intensity determine to
γ H2AX amount in fixed radiation sample.
It is used for the analysis of the quantitative biochip of phosphorus by LIBS
Fixed protein may then pass through LIBS analyses on kapton-epoxy resin holder, such as by swashing
Light microprobe device, and the XY mobile devices for rapidly and accurately moving holder are analyzed.Select what is used to disappear
Melt the wavelength of laser beam, closer to the wavelength for the compound for forming adduct：For described embodiment, using with
The yttrium pyralspite pulse laser of the neodymium-doped of the nanosecond pulse of 266 nano wave lengths and 4 millijoule energy/5, or Nd：YAG laser.
Two speculums can guide the surface of laser beam flying holder.In the beginning of its optical path, laser beam can pass through light
Then circle is focused on the surface of sample to be analysed by reflecting microscopical object lens.The position of sample can be used by CCD phases
Machine provide picture show and adjust by mobile device, the CCD camera is located above laser microprobe.Sample can lead to
Cross fixing device (such as passing through screw) positioning and be fixed in XY mobile devices.Mobile device can be grasped with 10 hertz of frequency
Make, described 10 hertz of frequency corresponds to the frequency of the Laser emission under the outside method of synchronization, in two continuous Laser emissions
Between with 30 microns of movements.
Analysis for plasma, optical fiber can be, for example, one end close to plasma is positioned over,
Light of the fiber guides from plasma to spectrometer entrance slit, the spectrometer equipped with pulse strengthen CCD camera (or
ICCD cameras).For example, spectral resolution can be equal to 0.032 for 100 microns of entrance slits in 410 nanometers.Mobile device
For example it can be controlled with ICCD cameras by suitable control device (such as being formed by single microcomputer).
Pulse laser and ICCD cameras can control via the control device of the whole analytical equipment of control.
Laser emission can be applied, and the light for for example extending to wave-length coverage from 248 nanometers to 258 nanometers can be obtained
Window is composed, the wave-length coverage digitizes around the optic spectrum line of intrinsic phosphorus (253.563 nanometers), and by the spectral window, so
Afterwards by the storage of suitable device in memory.Then control device can influence mobile device mobile example to new position
Put.
It can be advantageous to which pipe to be fixed to the fixed part of laser microprobe, and make it possible to be formed for producing gas
The device of (such as argon gas) is flowed, close to the surface of sample, produces plasma on to the sample.By this way, plasma
The amplitude of transmitting can increase, and the phenomenon of automatic absorption is reduced.
Phosphorus compose spectral line the intensity selected by 253.563 nanometers (IP253) be by subtract correspond to 254.120 nanometers and
The spectral background intensity of the average value of the intensity of 254.700 nano measurements is handled as sharp value.Target protein can pass through IP253
All sharp values of spectral line and quantified.
The spectrum of phosphorus and boron can be filtered with reliability factor Cdb, and the reliability factor Cdb is by following relation come really
It is fixed：
Cdb=[((IB-NB)-σN)/((IB-NB)+σN)] (1),
Wherein IBRepresent the intensity of boron, NBAnd σNThe base of about 249.084 nanometers and 250.363 nanometers of wavelength is represented respectively
The average value and standard deviation of plinth intensity, in all spectrally calculating.
Then phosphorus value of the lower relation of plane from selected quantitative spectrometric for target protein can be used：
Value P=∑s [(IP253-nP253)/(IB249-nB249)] (2)
Wherein IB249And IP253The intensity level of boron and phosphorus spectral line is represented respectively, and nP253 and nB249 are illustrated respectively in
The mean intensity of boron and phosphorus ambient background in given spectrum, corresponding to the scanning element by LIBS dielectric surfaces.
Claims (15)
1. a kind of be used for quantitative analysis method by plasma emission spectroscopy, the plasma is induced by laser beam
The plasma of at least one target (201) on biochip, it is characterised in that it includes the use of adjuvant (202),
The adjuvant, which allows to be formed, to be configured described dry with least one target (201) while the dry matrices (203) being ablated to
For dry matrix (203) to improve the analytical property of plasma, the adjuvant (202) has emission spectrum, the emission spectrum of adjuvant
The wavelength that spectral line has is different from the wavelength of the optic spectrum line of the quantitative analysis at least one target (201).
2. analysis method as claimed in claim 1, it is characterised in that at least one target (201) passes through at least one probe
(211) isolate on biochip so that probe-target is formed between each probe (211) and corresponding target (201) and is answered
Compound.
3. analysis method as claimed in claim 1, it is characterised in that the use of the adjuvant (202) allows amorphous structure
The formation of dry matrices (203).
4. analysis method as claimed in claim 1, it is characterised in that the use of the adjuvant (202) allows the dry of crystal structure
The formation of dry matrix (203).
5. analysis method as claimed in claim 2, it is characterised in that form the dry matrices (203) to cause the spy
Pin-target compound is located at the surface of holder (200), and all or part of by a number of dry matrices (203)
Ground encases.
6. analysis method as claimed in claim 2, it is characterised in that the dry matrices (203) and the probe (211) are solid
Determine to holder (200).
7. analysis method as claimed in claim 2, it is characterised in that form the adjuvant molecules direct combination of dry matrices (203)
To probe (211).
8. analysis method as claimed in claim 2, it is characterised in that the adjuvant molecules for forming dry matrices (203) are fixed to branch
Thing (200) is held, probe (211) is fixed to adjuvant molecules, and adjuvant molecules are fixed to holder (200).
9. analysis method as claimed in claim 2, it is characterised in that by holder (200) surface place one layer of adjuvant come
One layer of dry matrices (203) is formed, the dry matrices then probe (211) being placed in the support surface therefore formed
(203) on surface.
10. the analysis method as any one of preceding claims 1 to 4, it is characterised in that the dry matrices (203) by
Absorbing wavelength is formed close to the compound of laser beam wavelength, so as to which the wavelength that laser uses is included in the dry matrices
(203) in absorption spectrum.
11. the analysis method as any one of preceding claims 1 to 4, it is characterised in that the adjuvant (202) includes water
With the solution from sugared composition.
12. analysis method as claimed in claim 11, it is characterised in that the sugar is selected from polysaccharide and disaccharides.
13. the analysis method as any one of preceding claims 1 to 4, it is characterised in that each probe (211) formation
Standardization element (405) mark of internal standard.
14. analysis method as claimed in claim 11, it is characterised in that each probe (211) is by being grafted by standardization element
(405) mark.
15. analysis method as claimed in claim 11, it is characterised in that standardization element (405) is made up of boron.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR1258608 | 2012-09-13 | ||
| FR1258608A FR2995403B1 (en) | 2012-09-13 | 2012-09-13 | METHOD AND DEVICE FOR QUANTITATIVE MEASUREMENT BY LIBS OF BIOMOLECULAR TARGETS ON BIO-CHIP |
| PCT/EP2013/069072 WO2014041145A1 (en) | 2012-09-13 | 2013-09-13 | Method and device for the quantitative libs measurement of bio-molecular targets on a biochip |
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| Publication Number | Publication Date |
|---|---|
| CN104737001A CN104737001A (en) | 2015-06-24 |
| CN104737001B true CN104737001B (en) | 2017-11-10 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201380054861.1A Expired - Fee Related CN104737001B (en) | 2012-09-13 | 2013-09-13 | The method of the LIBS quantitative measurments of bio-molecular target on biochip |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US9400252B2 (en) |
| EP (1) | EP2895845A1 (en) |
| JP (1) | JP6220878B2 (en) |
| KR (1) | KR102165631B1 (en) |
| CN (1) | CN104737001B (en) |
| CA (1) | CA2884867C (en) |
| FR (1) | FR2995403B1 (en) |
| WO (1) | WO2014041145A1 (en) |
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| WO2016040924A1 (en) * | 2014-09-12 | 2016-03-17 | Purdue Research Foundation | Metal-antibody tagging and plasma-based detection |
| JP6296962B2 (en) * | 2014-11-12 | 2018-03-20 | 株式会社島津製作所 | Test body plate manufacturing method, test body plate manufactured thereby, and contained substance measuring apparatus using the same |
| EP3887804B1 (en) * | 2018-11-30 | 2023-05-03 | F. Hoffmann-La Roche AG | Laser induced breakdown spectroscopy for determining contaminants in lyophilised medications |
| CN113092395A (en) * | 2021-03-31 | 2021-07-09 | 四川大学 | Rapid cell classification and quantification method based on coffee ring |
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| WO2011006156A2 (en) * | 2009-07-10 | 2011-01-13 | University Of Florida Research Foundation, Inc. | Method and apparatus to laser ablation-laser induced breakdown spectroscopy |
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| WO2003025547A1 (en) * | 2001-09-21 | 2003-03-27 | Biomedlab Corporation | Method and device for screening analytes using surface plasmon resonance |
| US20060014212A1 (en) * | 2002-05-10 | 2006-01-19 | Epitome Biosystems, Inc. | Proteome epitope tags and methods of use thereof in protein modification analysis |
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| EP1632570A1 (en) * | 2003-05-16 | 2006-03-08 | Otsuka Pharmaceutical Co., Ltd. | Transcription chip |
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| FR2929011B1 (en) * | 2008-03-20 | 2013-01-04 | Commissariat Energie Atomique | METHOD AND DEVICE FOR HIGH QUANTITATIVE QUANTITATIVE MEASUREMENT OF BIOMOLECULAR TARGETS PRESENTED ON OR IN A BIOLOGICAL ANALYSIS MEDIUM. |
| JP2009288068A (en) * | 2008-05-29 | 2009-12-10 | Toshiba Corp | Analyzing method and analyzer |
| EP2281632B1 (en) * | 2009-07-02 | 2013-11-13 | Amic AB | Capillary driven assay device and its manufacture |
| FR2964458B1 (en) | 2010-09-06 | 2012-09-07 | Commissariat Energie Atomique | HIGH-RESOLUTION CARTOGRAPHY AND ANALYSIS DEVICE FOR ELEMENTS IN SOLIDS |
| EP2922861A4 (en) * | 2012-11-26 | 2016-09-14 | Caris Life Sciences Switzerland Holdings Gmbh | Biomarker compositions and methods |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008156491A2 (en) * | 2006-09-29 | 2008-12-24 | Zyomyx, Inc. | Devices and methods for analysis of samples with depletion of analyte content |
| JP2008256440A (en) * | 2007-04-03 | 2008-10-23 | Toshiba Corp | Analyzer |
| WO2010033452A2 (en) * | 2008-09-19 | 2010-03-25 | Delaware State University Foundation, Inc. | Mono-and multi-element coded libs assays and methods |
| WO2011006156A2 (en) * | 2009-07-10 | 2011-01-13 | University Of Florida Research Foundation, Inc. | Method and apparatus to laser ablation-laser induced breakdown spectroscopy |
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| US20150233837A1 (en) | 2015-08-20 |
| JP6220878B2 (en) | 2017-10-25 |
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| FR2995403A1 (en) | 2014-03-14 |
| US9400252B2 (en) | 2016-07-26 |
| WO2014041145A1 (en) | 2014-03-20 |
| FR2995403B1 (en) | 2014-09-12 |
| CA2884867C (en) | 2021-06-01 |
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